Protective-antigen (PA) based anthrax vaccines confer protection against inhalation anthrax by precluding the establishment of a systemic infection

ABSTRACT Systemic anthrax manifests as toxemia, rapidly disseminating septicemia, immune collapse, and death. Virulence factors include the anti-phagocytic γ-linked poly-d-glutamic acid (PGA) capsule and two binary toxins, complexes of protective antigen (PA) with lethal factor (LF) and edema factor. We report the characterization of LF, PA, and PGA levels during the course of inhalation anthrax in five rhesus macaques. We describe bacteremia, blood differentials, and detection of the PA gene (pagA) by PCR analysis of the blood as confirmation of infection. For four of five animals tested, LF exhibited a triphasic kinetic profile. LF levels (mean ± standard error [SE] between animals) were low at 24 h postchallenge (0.03 ± 1.82 ng/ml), increased at 48 h to 39.53 ± 0.12 ng/ml (phase 1), declined at 72 h to 13.31 ± 0.24 ng/ml (phase 2), and increased at 96 h (82.78 ± 2.01 ng/ml) and 120 h (185.12 ± 5.68 ng/ml; phase 3). The fifth animal had an extended phase 2. PGA levels were triphasic; they were nondetectable at 24 h, increased at 48 h (2,037 ± 2 ng/ml), declined at 72 h (14 ± 0.2 ng/ml), and then increased at 96 h (3,401 ± 8 ng/ml) and 120 h (6,004 ± 187 ng/ml). Bacteremia was also triphasic: positive at 48 h, negative at 72 h, and positive at euthanasia. Blood neutrophils increased from preexposure (34.4% ± 0.13%) to 48 h (75.6% ± 0.08%) and declined at 72 h (62.4% ± 0.05%). The 72-h declines may establish a “go/no go” turning point in infection, after which systemic bacteremia ensues and the host's condition deteriorates. This study emphasizes the value of LF detection as a tool for early diagnosis of inhalation anthrax before the onset of fulminant systemic infection.

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Efficacy of a Vaccine Based on Protective Antigen and Killed Spores against Experimental Inhalational Anthrax

Infection and Immunity ◽

10.1128/iai.01217-08 ◽

2008 ◽

Vol 77 (3) ◽

pp. 1197-1207 ◽

Cited By ~ 32

Author(s):

Yves P. Gauthier ◽

Jean-Nicolas Tournier ◽

Jean-Charles Paucod ◽

Jean-Philippe Corre ◽

Michèle Mock ◽

...

Keyword(s):

Guinea Pigs ◽

Protective Immunity ◽

Protective Antigen ◽

Virulent Strain ◽

Neutralizing Antibody ◽

Cutaneous Anthrax ◽

Anthrax Vaccines ◽

Mucosal Secretions ◽

Highly Virulent ◽

Lethal Doses

ABSTRACTProtective antigen (PA)-based anthrax vaccines acting on toxins are less effective than live attenuated vaccines, suggesting that additional antigens may contribute to protective immunity. Several reports indicate that capsule or spore-associated antigens may enhance the protection afforded by PA. Addition of formaldehyde-inactivated spores (FIS) to PA (PA-FIS) elicits total protection against cutaneous anthrax. Nevertheless, vaccines that are effective against cutaneous anthrax may not be so against inhalational anthrax. The aim of this work was to optimize immunization with PA-FIS and to assess vaccine efficacy against inhalational anthrax. We assessed the immune response to recombinant anthrax PA fromBacillus anthracis(rPA)-FIS administered by various immunization protocols and the protection provided to mice and guinea pigs infected through the respiratory route with spores of a virulent strain ofB. anthracis. Combined subcutaneous plus intranasal immunization of mice yielded a mucosal immunoglobulin G response to rPA that was more than 20 times higher than that in lung mucosal secretions after subcutaneous vaccination. The titers of toxin-neutralizing antibody and antispore antibody were also significantly higher: nine and eight times higher, respectively. The optimized immunization elicited total protection of mice intranasally infected with the virulentB. anthracisstrain 17JB. Guinea pigs were fully protected, both against an intranasal challenge with 100 50% lethal doses (LD50) and against an aerosol with 75 LD50of spores of the highly virulent strain 9602. Conversely, immunization with PA alone did not elicit protection. These results demonstrate that the association of PA and spores is very much more effective than PA alone against experimental inhalational anthrax.

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Identification of a Protein Subset of the Anthrax Spore Immunome in Humans Immunized with the Anthrax Vaccine Adsorbed Preparation

Infection and Immunity ◽

10.1128/iai.73.9.5685-5696.2005 ◽

2005 ◽

Vol 73 (9) ◽

pp. 5685-5696 ◽

Cited By ~ 28

Author(s):

Indira T. Kudva ◽

Robert W. Griffin ◽

Jeonifer M. Garren ◽

Stephen B. Calderwood ◽

Manohar John

Keyword(s):

Drug Targets ◽

Protective Antigen ◽

Hypothetical Protein ◽

The United States ◽

Surface Proteins ◽

Spore Surface ◽

Expression Library ◽

Anthrax Vaccine ◽

Anthrax Spore ◽

Anthrax Vaccines

ABSTRACTWe identified spore targets of Anthrax Vaccine Adsorbed (AVA)-induced immunity in humans by screening recombinant clones of a previously generated, limited genomicBacillus anthracisSterne (pXO1+, pXO2−) expression library of putative spore surface (spore-associated [SA]) proteins with pooled sera from human adults immunized with AVA (immune sera), the anthrax vaccine currently approved for use by humans in the United States. We identified 69 clones that reacted specifically with pooled immune sera but not with pooled sera obtained from the same individuals prior to immunization. Positive clones expressed proteins previously identified as localized on the anthrax spore surface, proteins highly expressed during spore germination, orthologs of proteins of diverse pathogens under investigation as drug targets, and orthologs of proteins contributing to the virulence of both gram-positive and gram-negative pathogens. Among the reactive clones identified by this immunological screen was one expressing a 15.2-kDa hypothetical protein encoded by a gene with no significant homology to sequences contained in databases. Further studies are required to define the subset of SA proteins identified in this study that contribute to the virulence of this pathogen. We hypothesize that optimal delivery of a subset of SA proteins identified by such studies to the immune system in combination with protective antigen (PA), the principal immunogen in AVA, might facilitate the development of defined, nonreactogenic, more-efficacious PA-based anthrax vaccines. Future studies might also facilitate the identification of SA proteins with potential to serve as targets for drug design, spore inactivation, or spore detection strategies.

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Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

Clinical and Vaccine Immunology ◽

10.1128/cvi.00046-13 ◽

2013 ◽

Vol 20 (7) ◽

pp. 986-997 ◽

Cited By ~ 2

Author(s):

Hang Lu ◽

Jason Catania ◽

Katalin Baranji ◽

Jie Feng ◽

Mili Gu ◽

...

Keyword(s):

Protective Antigen ◽

Lethal Factor ◽

Neutralization Assay ◽

Dominant Factor ◽

Anthrax Lethal Toxin ◽

Serum Samples ◽

Native Form ◽

Anthrax Lethal Factor ◽

Methionine Residues ◽

Anthrax Vaccines

ABSTRACTThe cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply.

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Comparability of ELISA and toxin neutralization to measure immunogenicity of Protective Antigen in mice, as part of a potency test for anthrax vaccines

Vaccine ◽

10.1016/j.vaccine.2009.05.045 ◽

2009 ◽

Vol 27 (33) ◽

pp. 4537-4542 ◽

Cited By ~ 11

Author(s):

P.M. Parreiras ◽

L.A. Sirota ◽

L.D. Wagner ◽

S.L. Menzies ◽

J.L. Arciniega

Keyword(s):

Protective Antigen ◽

Anthrax Vaccines

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Venezuelan Equine Encephalitis Virus-Vectored Vaccines Protect Mice against Anthrax Spore Challenge

Infection and Immunity ◽

10.1128/iai.71.3.1491-1496.2003 ◽

2003 ◽

Vol 71 (3) ◽

pp. 1491-1496 ◽

Cited By ~ 44

Author(s):

John S. Lee ◽

Angela G. Hadjipanayis ◽

Susan L. Welkos

Keyword(s):

Protective Antigen ◽

Vaccine Vector ◽

Mouse Strains ◽

Multiple Cloning Site ◽

Venezuelan Equine Encephalitis ◽

Equine Encephalitis Virus ◽

Primary Series ◽

Carboxy Terminal ◽

Anthrax Vaccines

ABSTRACT Anthrax, a disease usually associated with herbivores, is caused by the bacterium Bacillus anthracis. The current vaccine licensed for human use requires a six-dose primary series and yearly boosters and causes reactogenicity in up to 30% of vaccine recipients. A minimally reactogenic vaccine requiring fewer inoculations is warranted. Venezuelan equine encephalitis (VEE) virus has been configured for use as a vaccine vector for a wide variety of immunogens. The VEE vaccine vector is composed of a self-replicating RNA (replicon) containing all of the VEE virus nonstructural genes and a multiple-cloning site in place of the VEE structural genes. Four different anthrax vaccines were constructed by cloning the protective antigen (PA) gene from B. anthracis into the VEE vaccine vector. The anthrax vaccines were produced by assembling the vectors into propagation-deficient VEE replicon particles in vitro. A/J mice inoculated subcutaneously with three doses of the mature 83-kDa PA vaccine were completely protected from challenge with the Sterne strain of B. anthracis. Similar results were obtained with vaccines composed of the PA gene fused to either the B. anthracis secretory sequence or to a tissue plasminogen activator secretory sequence in three additional mouse strains. Mice were unprotected from challenge after inoculation with the carboxy-terminal 63-kDa PA vaccine. These results suggest that these VEE-vectored vaccines may be suitable as candidate vaccines against anthrax.

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Protective Antigen as a Correlative Marker for Anthrax in Animal Models

Infection and Immunity ◽

10.1128/iai.00792-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5871-5876 ◽

Cited By ~ 58

Author(s):

David Kobiler ◽

Shay Weiss ◽

Haim Levy ◽

Morly Fisher ◽

Adva Mechaly ◽

...

Keyword(s):

Animal Models ◽

Protective Antigen ◽

Therapeutic Window ◽

Bacterial Antigen ◽

Airborne Spores ◽

Efficient Treatment ◽

Time To Death ◽

Inhalation Anthrax ◽

Reliable Marker ◽

Time Required

ABSTRACT The most aggressive form of anthrax results from inhalation of airborne spores of Bacillus anthracis and usually progresses unnoticed in the early stages because of unspecific symptoms. The only reliable marker of anthrax is development of bacteremia, which increases with disease progress. Rapid diagnosis of anthrax is imperative for efficient treatment and cure. Herein we demonstrate that the presence and level of a bacterial antigen, the protective antigen (PA), a component of B. anthracis toxins, in host sera can serve as a reliable marker of infection. This was tested in two animal models of inhalation anthrax, rabbits and guinea pigs infected by intranasal instillation of Vollum spores. In both models, we demonstrated qualitative and quantitative correlations between levels of bacteremia and PA concentrations in the sera of sick animals. The average time to death in infected animals was about 16 h after the appearance of bacteremia, leaving a small therapeutic window. As the time required for immunodetection of PA can be very short, the use of this marker will be beneficial for faster diagnosis and treatment of inhalation anthrax.

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Human Monoclonal Antibody AVP-21D9 to Protective Antigen Reduces Dissemination of the Bacillus anthracis Ames Strain from the Lungs in a Rabbit Model

Infection and Immunity ◽

10.1128/iai.00352-07 ◽

2007 ◽

Vol 75 (7) ◽

pp. 3414-3424 ◽

Cited By ~ 47

Author(s):

Johnny W. Peterson ◽

Jason E. Comer ◽

Wallace B. Baze ◽

David M. Noffsinger ◽

Autumn Wenglikowski ◽

...

Keyword(s):

Monoclonal Antibody ◽

Bacillus Anthracis ◽

Protective Antigen ◽

Human Monoclonal Antibody ◽

Rabbit Model ◽

Concomitant Treatment ◽

Dependent Manner ◽

Lethal Challenge ◽

Tissue Sections ◽

Inhalation Anthrax

ABSTRACT Dutch-belted and New Zealand White rabbits were passively immunized with AVP-21D9, a human monoclonal antibody to protective antigen (PA), at the time of Bacillus anthracis spore challenge using either nasal instillation or aerosol challenge techniques. AVP-21D9 (10 mg/kg) completely protected both rabbit strains against lethal infection with Bacillus anthracis Ames spores, regardless of the inoculation method. Further, all but one of the passively immunized animals (23/24) were completely resistant to rechallenge with spores by either respiratory challenge method at 5 weeks after primary challenge. Analysis of the sera at 5 weeks after primary challenge showed that residual human anti-PA levels decreased by 85 to 95%, but low titers of rabbit-specific anti-PA titers were also measured. Both sources of anti-PA could have contributed to protection from rechallenge. In a subsequent study, bacteriological and histopathology analyses revealed that B. anthracis disseminated to the bloodstream in some naïve animals as early as 24 h postchallenge and increased in frequency with time. AVP-21D9 significantly reduced the dissemination of the bacteria to the bloodstream and to various organs following infection. Examination of tissue sections from infected control animals, stained with hematoxylin-eosin and the Gram stain, showed edema and/or hemorrhage in the lungs and the presence of bacteria in mediastinal lymph nodes, with necrosis and inflammation. Tissue sections from infected rabbits dosed with AVP-21D9 appeared comparable to corresponding tissues from uninfected animals despite lethal challenge with B. anthracis Ames spores. Concomitant treatment with AVP-21D9 at the time of challenge conferred complete protection in the rabbit inhalation anthrax model. Early treatment increased the efficacy progressively and in a dose-dependent manner. Thus, AVP-21D9 could offer an adjunct or alternative clinical treatment regimen against inhalation anthrax.

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Bacillus anthracis Edema Toxin Impairs Neutrophil Actin-Based Motility

Infection and Immunity ◽

10.1128/iai.00839-08 ◽

2009 ◽

Vol 77 (6) ◽

pp. 2455-2464 ◽

Cited By ~ 28

Author(s):

Sarah E. Szarowicz ◽

Russell L. During ◽

Wei Li ◽

Conrad P. Quinn ◽

Wei-Jen Tang ◽

...

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Fold Increase ◽

Polymorphonuclear Neutrophil ◽

Actin Assembly ◽

Anthrax Lethal Toxin ◽

Edema Factor ◽

Low Concentrations ◽

Inhalation Anthrax ◽

Edema Toxin

ABSTRACT Inhalation anthrax results in high-grade bacteremia and is accompanied by a delay in the rise of the peripheral polymorphonuclear neutrophil (PMN) count and a paucity of PMNs in the infected pleural fluid and mediastinum. Edema toxin (ET) is one of the major Bacillus anthracis virulence factors and consists of the adenylate cyclase edema factor (EF) and protective antigen (PA). Relatively low concentrations of ET (100 to 500 ng/ml of PA and EF) significantly impair human PMN chemokinesis, chemotaxis, and ability to polarize. These changes are accompanied by a reduction in chemoattractant-stimulated PMN actin assembly. ET also causes a significant decrease in Listeria monocytogenes intracellular actin-based motility within HeLa cells. These defects in actin assembly are accompanied by a >50-fold increase in intracellular cyclic AMP and a >4-fold increase in the phosphorylation of protein kinase A. We have previously shown that anthrax lethal toxin (LT) also impairs neutrophil actin-based motility (R. L. During, W. Li, B. Hao, J. M. Koenig, D. S. Stephens, C. P. Quinn, and F. S. Southwick, J. Infect. Dis. 192:837-845, 2005), and we now find that LT combined with ET causes an additive inhibition of PMN chemokinesis, polarization, chemotaxis, and FMLP (N-formyl-met-leu-phe)-induced actin assembly. We conclude that ET alone or combined with LT impairs PMN actin assembly, resulting in paralysis of PMN chemotaxis.

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Use of Site-Directed Mutagenesis To Model the Effects of Spontaneous Deamidation on the Immunogenicity of Bacillus anthracis Protective Antigen

Infection and Immunity ◽

10.1128/iai.00863-12 ◽

2012 ◽

Vol 81 (1) ◽

pp. 278-284 ◽

Cited By ~ 15

Author(s):

Anita Verma ◽

Beth McNichol ◽

Rocío I. Domínguez-Castillo ◽

Juan C. Amador-Molina ◽

Juan L. Arciniega ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Large Scale ◽

Protective Antigen ◽

Cd Spectroscopy ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Vaccine Formulation ◽

Wild Type ◽

Vaccine Immunogenicity ◽

Anthrax Vaccines

Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled for large-scale use in an emergency situation; however, spontaneous deamidation of purified vaccine antigens has the potential to adversely affect vaccine immunogenicity over time. In order to explore whether spontaneous deamidation of recombinant protective antigen (rPA)—the major component of new-generation anthrax vaccines—affects vaccine immunogenicity, we created a “genetically deamidated” form of rPA using site-directed mutagenesis to replace six deamidation-prone asparagine residues, at positions 408, 466, 537, 601, 713, and 719, with either aspartate, glutamine, or alanine residues. We found that the structure of the six-Asp mutant rPA was not significantly altered relative to that of the wild-type protein as assessed by circular dichroism (CD) spectroscopy and biological activity. In contrast, immunogenicity of aluminum-adjuvanted six-Asp mutant rPA, as measured by induction of toxin-neutralizing antibodies, was significantly lower than that of the corresponding wild-type rPA vaccine formulation. The six-Gln and six-Ala mutants also exhibited lower immunogenicity than the wild type. While the wild-type rPA vaccine formulation exhibited a high level of immunogenicity initially, its immunogenicity declined significantly upon storage at 25°C for 4 weeks. In contrast, the immunogenicity of the six-Asp mutant rPA vaccine formulation was low initially but did not change significantly upon storage. Taken together, results from this study suggest that spontaneous deamidation of asparagine residues predicted to occur during storage of rPA vaccines would adversely affect vaccine immunogenicity and therefore the storage life of vaccines.

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